Instant Notes: Analytical Chemistry

Rfvalues, e.g. amino acids. A single sample is spotted close to one corner of

the plate and, after development, there is a partial separation of the solutes

along one edge. The plate is dried, turned through 90∞and developed a

second time, but using a different mobile phase composition for which the

solutes have different distribution ratios compared to those for the first

mobile phase. This results in the partially-separated solutes separating

further because of changes to their migration rates. Visualization gives a 2-D

mapor fingerprintof the sample components. The procedure for the separa-

tion of 14 amino acids is shown diagrammatically in Figure 2.

● High-performance plates (HPTLC). These are coated with a thinner layer

(100mm thick) of a 5mm particle diameter stationary phase with a very

narrow range of sizes. Sensitivity and resolution are improved because solute

spots are compact, development is much faster, partly because smaller plates

can be used, and solvent consumption is reduced. HPTLCplates with silica

and bonded-phase silicas are commercially available.

TLCis applicable to a very wide range of mainly organic solutes. It is used

primarily in the biochemical, pharmaceutical, clinical and forensic areas for

qualitative analysisby the comparison of Rfvalues of sample solutes with those

of standards run on the same plate. It is especially useful for checks on purity, to

monitor the course of reactions and production processes, and to characterize

complex materials such as natural products. The screening of samples for drugs

Applications of
TLC

D3 – Thin-layer chromatography 135

Solvent 1 n-butanol/acetic

acid/water (12:3:5)

Sample

application

Solvent 2 Phenol/H 2 O

GLU

GLYTHRALA

TA U

SER LY SARG

PRO-OHPRO

a-ABA

VAL Ph-ALA

LEU

a bab

a

b

a

1st b

solvent

Solvent front 2nd

solvent

Solvent front

Sample Origin Turn 90 Origin

After 1st development After 2nd development

Fig. 2. Two-dimensional TLC of a mixture of 14 amino acids. Top panel, reproduced from R.J. Hamilton & S. Hamilton,
Thin-Layer Chromatography, 1987. © John Wiley & Sons Ltd. Reproduced with permission. Bottom panel, reproduced from
A. Braithwaite & F.J. Smith, Chromatographic Methods, 5th edn, 1996, first published by Blackie Academic & Professional.